Aminoalkylphosphotriester oligonucleotide derivatives

ABSTRACT

Disclosed are methods of producing a synthetic oligonucleotide of selected nucleotide sequence which is internally functionalized at least one selected location with an aminoalkylphosphotriester, and labelled with a nonradioactive material. Also disclosed are oligonucleotides produced by this method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of Ser. No. 08/206,175, filed Mar. 3,1994, which is a continuation-in-part of applicant's application Ser.No. 07/490,481, entitled "SITE-SPECIFIC FUNCTIONALIZATION OFOLIGODEOXYNUCLEOTIDES FOR NON-RADIOACTIVE LABELLING", filed Mar. 8, 1990(now U.S. Pat. No. 5,321,131, issued Jun. 14, 1994).

FUNDING

Work described herein was supported by Cooperative Grant U01 124846 fromthe National Institute of Allergies and Infectious Disease and a grantfrom the G. Harold and Leila Y. Mathers Foundation.

BACKGROUND OF THE INVENTION

There is at present growing interest in non-radioactively labelledmodified oligode. oxynucleotides. Biotin (Agrawal et al. (1986) Nucl.Acids Res. 14:6227-6245; Agrawal (1989) Tet. Lett. 30: 7025-7028),florophores (Cardullo et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:8790-8794; Agrawal et al. (1988) J. Cell Biology 107:468; Haralambidiset 0al. (1989) Nucl. Acids Res. 18(3) :501-505), intercalating (Heleneet al. Oligodeoxynucleotides--Antisense Inhibitors of Gene Expression(Cohen, ed.) Macmillan Press (1989) pp.137-166) and chelating reagents(Oser et al. (1988) Nucl. Acids Res. 16:1181-1196) attached to syntheticoligonucleotides are becoming important tools of molecular biology. Avariety of enzymatic and chemical procedures have been developed fortheir synthesis (Matthews et al. (1988) Anal. Biochem. 169: 1-25).Central to some of these procedures are (a) the introduction of areactive group at either the 3'- or 5'- terminus of the oligonucleotide(Agrawal et al. (1986) Nucl. Acids Res. 14:6227-6245; Agrawal (1989)Tet. Lett. 30:7025-7028; Fidanza et al. (1989) J. Am. Chem. Soc. 111:9117-9119; Nelson (1989) Nucl. Acids Res. 17:7187-7194 or (b) thesynthesis of modified nucleosides which contain the masked reactivegroup and are incorporated into the nucleic acid (Fidanza et al. (1989)J. Am. Chem. Soc. 111: 9117-9119). The presently-available methods areuseful, but are limited in their usefulness for site specific internalnon-radioactive labelling of synthetic oligonucleotides.

SUMMARY OF THE INVENTION

The present invention relates to compounds consisting of a plurality ofnucleosides including adenosine, thymidine, cytidine, guanosine, anduridine, which are covalently linked by at least oneaminoalkylphosphoramidate, aminoalkylphosphotriester, oraminoalkylphosphorothioamidate residue. As used herein the terms"adenosine," "thymidine," "cytodine," and "guanosine" are intended toinclude the ribose and deoxyribose forms of these nucleosides.

The present invention also relates to a method of site specificfunctionalization of oligonucleotides of any sequence, length, or havingany internucleotide linkage for non-radioactive labelling, as well as tothe functionalized oligonucleotides and non-radioactively labelledoligonucleotides produced by the method. This method makes it possibleto modify one or more selected internucleoside phosphate (s) in asynthetic oligonucleotide in such a manner that it (they) can be used toincorporate a non-radioactive material into the molecule. In particular,the method of the present invention is used to modify one or moreselected internucleoside phosphates in a synthetic oligodeoxynucleotide,oligoribonucleotide, or oligonucleotide containing bothdeoxyribonucleotides and ribonucleotides, to giveaminoalkylphosphoramidate, aminoalkylphosphotriester, oraminoalkylphosphorothioamidate residues. The amino group(s) of theresulting modified residue(s) then further react with a non-radioactivelabel, such as biotin, fluorescein, or rhodamine (e.g.,N-hydroxysuccinimide ester of biotin, N-caproyl amidobiotin, and avariety of fluorophore isothiocyantes), or derivatives thereof, toproduce a non-radioactively labelled oligonucleotide in which the labelis present at a predetermined location or locations.

In one embodiment the present method, an H-phosphonate internucleosidelinkage is oxidized with an appropriately protected diamine, such asN-1-trifluoroacetylhexanediamine (CF₃ CONH(CH₂) ₆ NH₂),N-1-trifluoroacetylpentanediamine (CF₃ CONH (CH₂) ₅ NH₂),N-1-trifluoroacetylbutyldiamine (CF₃ CONH(CH₂)₄ NH₂),N-1-trifluoroacetylpropyldiamine (CF₃ CONH(CH₂)₃ NH₂), orN-1-trifluoroacetylethyldiamine (CF₃ CONH(CH₂)₂ NH₂), in the presence ofan appropriate solvent, such as carbon tetrachloride, to give aphosphoramidate or phosphorothioamidate internucleoside linkage(Zwierzak (1975) Synthesis pp. 507-508; Froehler et al. (1989) Nucl.Acids Res. 16: 4831-4839; Letsinger et al. (1988) J. Am. Chem. Soc.110:4470-4471; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA)85:7079-7083; Jager et al. (1988) Biochem. 27: 7237-7246).Alternatively, the H-phosphonate internucleoside linkage is oxidized togive a phosphotriester internucleoside linkage with an appropriatelyprotected aminoalkyl alcohol inN-methylimidazole-triethylamine-carbontetrachloride. The resultingphosphoramidate, phosphotriester, or phosphorothioamidateinternucleoside linkage is stable under oligonucleotide assemblyconditions using phosphoramidite chemistry (Caruthers et al. (1987)Meth. Enzymol. 154:287-313) and subsequent deprotection steps.

The methods of the invention are used to produce non-radioactivelylabelled oligonucleotides which include a non-radioactive material atone or more sites and are useful in research and in the diagnosis andtreatment of diseases and conditions of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which:

FIG. 1A is a graphic representation of the results of analytical ionexchange HPLC of oligomers 1 (chromatograph (a)), 2 (chromatograph (b)),and 5 (chromatograph (c ));

FIG. 1B is a graphic representation of the results of reversed phaseHPLC of ion exchange purified oligomers 1 (chromatograph (d)), 2(chromatograph (e)), and 3 (chromatograph (f));

FIG. 2A is a graphic representation of the results of reversed phaseHPLC traces of oligomer 2 (chromatogram (a)), and reaction mixture ofoligomer with biotin N-hydroxysuccinimide (chromatograph (b)); and

FIG. 2B is a graphic representation of the results of reversed phaseHPLC traces of oligomer 5 (chromatograph (c)) and reaction mixture ofoligomer 5 with biotin N-hydroxysuccinimide (chromatograph (d)).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. The issuedU.S. patent and allowed applications cited herein are herebyincorporated by reference.

The present invention relates to a method of producing oligonucleotideswhich have a desired (selected) nucleotide sequence and which arelabelled internally with a non-radioactive material or reporter group atone or more internucleoside linkages. In the method of the presentinvention, one or more selected internucleoside phosphate residues aremodified to produce aminoalkylphosphoramidate,aminoalkylphosphotriester, or aminoalkylphosphorothioamidate residueswhich are present in an oligonucleotide at all or any number of selectedpositions. The amino group(s) in such modified (i.e., functionalized)residues is further reacted with a label or reporter group, resulting inproduction of a non-radioactively labelled oligonucleotide labelledinternally at selected location(s).

Briefly, the present method is carried out by oxidizing an H-phosphonateinternucleoside linkage using an appropriately protected diamine oramino alcohol represented by the formulae YNH (CH₂)_(n) NH₂, YNH(CH₂)OH,in which "Y" is a base labile protecting group and "n" can be 2 or more.For example, an H-phosphate internucleoside is oxidized usingN-1-trifluoroacetylethyldiamine (CF₃ CONH(CH₂)₂ NH₂),N-1-trifluoroacetylpropyldiamine (CF₃ CONH(CH₂)₃ NH₂),N-1-trifluoroacetylbutyldiamine (CF₃ CONH(CH₂)₄ NH₂),N-1-trifluoroacetylpentanediamine (CF₃ CONH(CH₂)₅ NH₂), orN-1-trifluoracetylhexanediamine pentane (CF₃ CONH(CH₂)₆ NH₂), in thepresence of an appropriate solvent such as anhydrous carbontetrachloride. As a result, a primary aliphatic amine is incorporated atthe internucleoside phosphate as a phosphoramidate.

In the case of phosphotriester linkages, oxidation is carried out usinga suitably protected amino alcohol, represented by the formulaYNH(CH₂)_(n) OH, in which "Y" is a base labile protecting group and "n"can be 2 or more. For example, an H-phosphonate internucleoside linkageis oxidized using FMOC--NH (CH₂)₂ OH, FMOC--NH(CH₂)₃ OH, FMOC--NH(CH₂)₄OH, FMOC--NH(CH₂)₅ OH, or N-1-fluoroenylmethyoxcarbonylaminohexanol(pentanol) (FMOC--NH(CH₂)₆ OH) in the presence ofN-methylimidazole-triethylamine-carbon tetrachloride (at, e.g., 5:5:90).

In the case of aminoalkylphosphorothioamidate linkages, oxidation iscarried out using a suitably protected diamine as described above.

The remaining nucleotides needed to produce the desired nucleotidesequence are covalently added using art-recognized techniques, such asphosphoramidite, H-phosphonate chemistry, or methyl phosphoramiditechemistry (see, e.g., Uhlmann et al. (1990) Chem. Rev. 90:543-584;Agrawal et al. (1987) Tet. Lett. 28: (31) :3539-3542); Caruthers et al.(1987) Meth. Enzymol. 154: 287-313; copending U. S. patent applicationSer. No. 07/334,679 ("METHOD OF SYNTHESIZING OLIGONUCLEOTIDES AND THEIRANALOGS ADAPTABLE TO LARGE SCALE SYNTHESIS," by S. Agrawal and P.Zamecnik, filed Apr. 6, 1989). After the desired oligonucleotide isproduced, the protecting group present on the primary aliphatic amine oramino alcohol is removed. The unmasked amino group can now react withone or more selected labels or reporter groups. As a result, theoligonucleotide is non-radioactively labelled at one or more selectedinternal locations. One or both amino groups present in the diaminereact with the selected label.

The methods of the present invention are represented in three sets ofsteps below. The following is an explanation of those steps, withreference to the respective reactants and steps represented below.##STR1##

In Step 1 of the three methods above, initial coupling of twonucleotides (designated Nu₁ and Nu₂) is carried out using H-phosphonatechemistry. Generally, Nu₁ is bound to a solid support, such as CPG, andterminates in a dimethoxytrityl residue (designated (I)). As a result, asupport-bound dinucleoside H-phosphonate (II) is produced.

In Step 2, the support-bound dinucleoside H-phosphonate (II) issubsequently oxidized by being combined with an appropriately protecteddiamine or amino alcohol, in the presence of a suitable solvent,resulting in formation of a phosphoramidate, phosphotriester, orphosphorothioamidate internucleoside linkage and linking of theprotected diamine or aminoalcohol, to the dinucleoside through theunprotected amino group of the diamine or amino alcohol. The resultingproduct is designated (III).

Then, in Step 3, the dimethoxytrityl residue present on the unbound endof (I) is removed and the remaining nucleotides (e.g., Nu₃, Nu₄, and soon up to Nu_(n)) of the desired oligonucleotide to be produced arecovalently added at the now free end, using phosphoramidite,H-phosphonate, or some other chemistry, producing a support-boundoligonucleotide (IV) which includes the phosphoramidate,phosphotriester, or phosphorothioamidate linkage produced in Step 2.

In Step 4, the protecting group "Y" present on the diamine is removedand the compound is removed from the solid support. This results inproduction of an unbound functionalized oligonucleotide (V) (i.e., anaminoaliphatic oligomer or an oligonucleotide having a desirednucleotide sequence and an alkylamino group present at the selectedinternucleoside phosphate(s) as a phosphoramidate, phosphotriester, orphosphorothioamidate.

In Step 5, the unbound functionalized oligonucleotide is reacted with anappropriate form of a non-radioactive material, which becomes bound tothe amino group and serves as a label or reporter group on theoligonucleotide. This results in production of an oligonucleotidelabelled site specifically with a non-radioactive material. Thenon-radioactive material can be a fluorophore, chemiluminescent tag,spin label, enzyme, chelator, heterocyclic molecule, protein, lipid,drug derivative, antigen, an intercalator, or other organic or inorganicmoiety.

It is possible, using the present method, to produce oligonucleotides ofdesired sequence which are labelled internally at one or morenucleosides. The oligonucleotide backbone can be unmodified (e.g., as itoccurs in nature, i.e., phosphodiester) or modified (e.g., amidate,alkylphosphate, phosphothioate, phosphodithioate, phosphate ester,phosphotriester, carbamate, carboxy methyl ester, or carbonateacetamidate containing backbones). The label present at two or moresites can be the same (e.g., biotin) or different, and can be present atas many sites as desired. As described in the EXAMPLES below, anoligomer of any sequence can be produced, functionalized at a selectedsite or sites, and labelled at the site(s) with a non-radioactivematerial.

In the above description of the present method, two single nucleotides(designated Nu₁ and Nu₂) are initially joined using H-phosphonatechemistry and the resulting oligonucleotide is functionalized at theinternucleoside phosphate linkage formed between Nu₁ and Nu₂. However,any number of nucleotides can be joined, using art-recognized techniquessuch as H-phosphonate chemistry, before modification of a selectedinternucleoside phosphate linkage is carried out.

For example, a 5mer, 17mer, 25mer, and 50mer functionalized at a singleinternal site, at two internal sites, at three contiguous ornon-contiguous internal sites, and at multiple non-contiguous andcontiguous sites can be produced by the method of the invention. UsingH-phosphonate chemistry, for example, an internal nucleotide 1 (e.g., asupport-bound nucleotide such as Nu₁) can be added to, resulting inproduction of a longer sequence (e.g., Nu₁₀ Nu₉ . . . Nu₁). The longersequence can then be functionalized by the method described above,resulting in production of a functionalized oligonucleotide (e.g., Nu₁₀↓Nu₉ . . . Nu₁, in which the internucleoside phosphate linkage betweenNu₉ and Nu₁₀ is modified). The modified oligonucleotide can then befurther elongated by addition of selected nucleotides to produce amodified oligonucleotide of desired sequence (e.g., Nu_(n) . . . Nu₁₀,↓Nu₉ . . . Nu₁).

The protecting group present can be removed as described above.Alternatively, the functionalized oligonucleotide initially produced canbe extended (by addition of selected nucleotides), and one or moreadditional internucleoside phosphate linkages can be modified, therebyproducing a functionalized oligonucleotide which has two or more sitesat which non-radioactive material can be added.

Protecting groups other than trifluoroacetyl (--CO--CF₃), as describedabove, can also be used to protect the diamine or amino alcohol. Otherbase labile protecting groups, such as FMOC and TBOC, may also be used.The linker present between the two amino groups in the diamine used canbe of any suitable length (e.g., --(CH₂)₂ -- to --(CH₂)_(n) --),preferably from about 2 to about 6 carbon atoms; the length used in aparticular case can be determined empirically. The diamine can bebranched or unbranched and bi-functional or multifunctional.

Assessment of the functionalized oligomers made by the method of theinvention can be carried out, for example, by analytical highperformance liquid chromatography (HPLC), reversed phase HPLC, and highperformance capillary electrophoresis (HPCE).

The following examples illustrate the preferred modes of making andpracticing the present invention, but are not meant to limit the scopeof the invention since alternative methods may be utilized to obtainsimilar results.

EXAMPLES

1. Preparation of Oxidizing Agents

N-1-trifluoroacetylhexanediamine, (CF₃ CONH(CH₂)₆ NH₂) is prepared byadding ethyltrifluoroacetate (1.2 ml, 10 mmol) dropwise over one hour toa stirred mixture of hexanediamine (1.16 g; 10 mmol) and triethylamine(1 ml; 7 mmol) in 20 ml methanol. The solution is stirred overnight.After removal of solvents, the reaction mixture is flash chromatographedon silica using 0-25% methanol in dichloromethane. The fractionscontaining the desired product are pooled and concentrated to give acolorless powder (1.1 gm, yield-42.6%); melting point 52° C; ¹ H NMR(CDCl₃, d, TMS=) .00) 7.1-7.2 (m, 3H, NH₂, NH) 3.2-3.3 (m, 2H,CO--NH--CH₂), 2.8-2.9 (m 2 H, CH₂ --NH₂) 1.2-1.6 (m, 8H,--CH--(CH₂)₂--CH₂ --).

N-1-trifluoroacetylethyldiamine (CF₃ CONH(CH₂)₂ NH₂ is prepared byadding ethyltrifluoroacetate (1.2 ml, 10 mmol) dropwise over one hour toa stirred mixture of ethylenediamine (0.6 g; 10 mmol) and triethylamine(1 ml; 7 mmol) in 20 ml methanol. The solution is stirred overnight.After removal of solvents, the reaction mixture is flash chromatographedon silica using 0-25% methanol in dichloromethane. The fractionscontaining the desired product are pooled and concentrated to give acolorless product.

N-1-trifluoroacetylpropyldiamine (CFCONH(CH₂)₃ NH₂) is prepared byadding ethyltrifluoroacetate (1.2 ml, 10 mmol) dropwise over one hour toa stirred mixture of 1,3-diaminopropane (0.74 g; 10 mmol) andtriethylamine (1 ml; 7 mmol) in 20 ml methanol. The solution is stirredovernight. After removal of solvents, the reaction mixture is flashchromatographed on silica using 0-25% methanol in dichloromethane. Thefractions containing the desired product are pooled and concentrated togive a colorless product.

N-1-trifluoroacetylbutyldiamine (CFCONH(CH₂)₄ NH₂) is prepared by addingethyltrifluoroacetate (1.2 ml, 10 mmol ) dropwise over one hour to astirred mixture of 1,4-diaminobutane (0.88 g; 10 mmol) and triethylamine(1 ml; 7 mmol) in 20 ml methanol. The solution is stirred overnight.After removal of solvents, the reaction mixture is flash chromatographedon silica using 0-25% methanol in dichloromethane. The fractionscontaining the desired product are pooled and concentrated to give acolorless product.

N-1-trifluoroacetylpentanediamine (CFCONH(CH₂)₅ NH₃) is prepared byadding ethyltrifluoroacetate (1.2 ml, 10 mmol) dropwise over one hour toa stirred mixture of 1,5-diaminopentane (1.02 g; 10 mmol) andtriethylamine (1 ml; 7 mmol) in 20 ml methanol. The solution is stirredovernight. After removal of solvents, the reaction mixture is flashchromatographed on silica using 0-25% methanol in dichloromethane. Thefractions containing the desired product are pooled and concentrated togive a colorless product.

2. Preparation of Oligonucleotides to be Functionalized

Three 5mers, three 17mers, three 25mers, and three 50mers, each having adifferent nucleotide sequence, are prepared using H-phosphonatechemistry as described in Uhlmann et al. (Chem. Rev. (1990) 90:534-583).These oligomers are listed in TABLE 1.

                                      TABLE 1                                     __________________________________________________________________________                                               SEQ ID                             SEQUENCE                                   NO:                                __________________________________________________________________________    AAATG                                      1                                  CGGCA                                      2                                  UGCCA                                      3                                  GTAAAACGACGGCCAGT                          4                                  GTATTCAAAGGAGTACC                          5                                  GAGCAUCACGGUGAGCG                          6                                  CTCTCGCACCCATCTCTCTCCTTCT                  7                                  GAATGACTGATTGAGTGACTGAATG                  8                                  CAGUGACUGACUGAGCGACUGAACU                  9                                  CGATGCGTAGGCCTAGAACTGGACTTACACTGGGATGCAAGTCAGCTGAT                                                                       10                                 TCAGTCGAGAGCTCGAGCTCGAGCGCTAGCGATGCAGTGCGATCGATAGC                                                                       11                                 AGCGCACUAGGCAAGUCCGCGAGAUCCACGAGCCAAUACGGACUCGCAGA                                                                       12                                 __________________________________________________________________________

These same oligomers are also prepared with, in addition to theaminoalkyltriester, aminoalkylphosphoramidate, and/oraminoalkylphosphorothioamidate linkages, at least one nonfunctionalized,nonphosphodiester internucleotide linkages including a phosphorothioate,phosphorodithioate, phosphoramidate, alkylphosphonate,alkylphosphorothioate, and carbamate linkages at selected positionsusing the procedures described in Uhlmann et al. Chem. Rev. (1990)90:534-583).

03. Functionalization

The following steps are carried out to produce a non-radioactivelylabelled oligonucleotide with a functionalized phosphoramidate:##STR2## 1. Initially, thymidine (T) (Nu₁) and protected deoxyguanosineH-phosphonate (G) (Nu₂) (I) are coupled using art-recognizedH-phosphonate chemistry, resulting in production of a support-bounddinucleoside H-phosphonate (II).

2. The support-bound dinucleoside H-phosphonate (II) is oxidized using4% N-1-trifluoroacetyldiaminohexane (NH₂ (CH₂)₆ NH--CO--CF₃) in carbontetrachloride-dioxane (8:2, volume/volume) for 30 minutes, resulting information of a phosphoramidate internucleoside linkage between Nu₂ andNu₁ (III). In other tests, oxidation is carried out using CF₃ CONH(CH₂)₂NH₂, CF₃ CONH(CH₂)₃ NH₂, CF₃ CONH(CH₂)₄ NH₂, or CF₃ CONH (CH₂)₅ NH₂, inanhydrous carbon tetrachloride.

3. The remainder of the nucleotide sequence of the oligonucleotide isproduced in a two-step procedure in which the dimethoxytrityl residue[DMTr] is removed from the guanidine nucleotide now bound to the solidsupport (Nu₂ in the reaction scheme above) and the desired nucleotides A(Nu₃), C (Nu₄), C(Nu₅) and so on up to G (Nu₁₇) (SEQ ID NO:4) are addedstepwise (i.e., to the now free end of the dinucleoside which, forconvenience, can be referred to as the 5' end using phosphoramidite orH-phosphonate chemistry resulting in molecule (IV).

4. The protecting group (CO--CF₃) present on NH(CH₂)₆ NH--CO--CF₃ isremoved during deprotection of oligonucleotides in aqueous ammonia for 6hours at 55° C., resulting in production of a functionalizedoligonucleotide (V) (in which the previously protected amino group isunprotected) of the desired sequence, in which there is anaminoalkylphosphoramidate residue of the formula: ##STR3## present atthe desired internucleoside phosphate linkage(s). 5. The unboundmodified oligonucleotide with the aminoalkylphosphoramidate residue isreacted with a non-radioactive material, such as biotin, fluorescein orrhodamine in appropriate form (e.g., N-hydroxysuccinimide ester ofbiotin, N-caproyl amidobiotin, fluorophore isothiocyanates), whichbecomes bound to the amino group of the aminoalkylphosphoramidateinternucleoside linkage.

Alternatively, the following steps are carried out to produce anon-radioactively labelled oligonucleotide with a functionalizedaminoalkylphosphotriester: ##STR4## 1. Initially, T (Nu₁) and G (Nu₂)are coupled as described in Uhlmann et al. (Chem. Rev. (1990)90:551-552).

2. Oxidation is carried out using a protected amino alcohol such asFMOC--NH(CH₂)₂ OH, FMOC--NH(CH₂)₃ OH, FMOC--NH(CH₂)₄ OH, FMOC--NH(CH₂)₅OH, or N-1-fluoroenylmethyoxcarbonylamino-hexanol (pentanol)(FMOC--NH(CH₂)₆ OH) in the presence ofN-methylimidazole-triethylamine-carbontetrachloride, resulting in theformation of a phosphotriester linkage between G and T.

3. The remainder of the nucleotide sequence of the oligonucleotide iscovalently added using phosphoramidite, H-phosphonate, ormethylphosphonoamidite chemistry as described by copending patentapplication entitled "METHOD OF SYNTHESIZING OLIGONUCLEOTIDES AND THEIRANALOGS ADAPTABLE TO LARGE SCALE SYNTHESIS" (Ser. No. 07/334,679) andUhlmann et al. (1990) Chem. Rev. 90:534-583).

4. After the oligonucleotide is produced, the protecting group on theprimary aliphatic amino alcohol is removed, resulting in the productionof functionalized oligonucleotide in which there is anaminoalkylphosphotriester residue present at the desired internucleosidephosphotriester linkage(s).

5. The unbound modified oligonucleotide with theaminoalkylphosphotriester residue in then reacted with biotin,fluoroscein, rhodamine, or derivatives thereof, or with any othernon-radioactive labels capable of being bound to the amino group of theresidue.

To prepare an oligonucleotide with a functionalizedaminoalkylphosphorothioamidate, the method is carried out as follows:##STR5## 1. Initially, thymidine (Nu₁) and guanidine (Nu₂) (I) arecoupled using art-recognized H-phosphonate chemistry, resulting inproduction of a support-bound dinucleoside H-phosphonate (II).

2. The support-bound dinucleoside H-phosphonate (II) is oxidized using4% N-1-trifluoroacetyldiaminohexane (NH(CH₂)₆ NH--CO--CF₃) in carbontetrachloride-dioxane (8:2, volume/volume) for 30 minutes, resulting information of a phosphonothioate internucleoside linkage between Nu₂ andNu₁ (III). In other tests, oxidation is carried out using CF₃ CONH(CH₂)₂NH₂, CF₃ CONH(CH₂)₃ NH₂, CF₃ CONH(CH₂)₄ NH₂, or CF₃ CONH(CH₂)₅ NH₂ inanhydrous carbon tetrachloride.

3. The remainder of the nucleotide sequence of the oligonucleotide isproduced in a two-step procedure in which the dimethoxytrityl residue[DMTr] is removed from the guanidine nucleotide now bound to the solidsupport (Nu₂ in the reaction scheme above) and the desired nucleotides A(Nu₃), C (Nu₄), C(Nu₅) and so on up to G (Nu₇) (SEQ ID NO:4) are addedstepwise (i. e., to the now free end of the dinucleoside which, forconvenience, can be referred to as the 5' end using phosphoramidite orH-phosphonate chemistry resulting in molecule (IV).

4. The protecting group (CO--CF₃) present on NH(CH₂)₆ NH--CO--CF₃ isremoved during deprotection of oligonucleotides in aqueous ammonia for 6hours at 55° C., resulting in production of a functionalizedoligonucleotide (V) (in which the previously protected amino group isunprotected) of the desired sequence, in which there is anaminoalkylphosphorothioamidate residue of the formula: ##STR6## presentat the desired internucleoside phosphate linkage(s). 5. The unboundmodified oligonucleotide with the aminoalkylphosphorothioamidate residueis reacted with a non-radioactive material, such as biotin, fluoresceinor rhodamine in appropriate form (e.g., N-hydroxysuccinimide ester ofbiotin, N-caproyl amidobiotin, fluorophore isothiocyanates), whichbecomes bound to the amino group of the aminoalkylphosphonothioateinternucleoside linkage.

Other functionalized oligomers with 5, 17, 25, and 50 nucleotides arealso prepared. All of these oligonucleotides are listed in TABLES 2A-2Dbelow, where "↓" indicates the position of the functionalized residue.

                  TABLE 2A                                                        ______________________________________                                                                   SEQ ID                                             OLIGOMER #     SEQUENCE    NO:                                                ______________________________________                                        1              AAATG       1                                                  2              AAA.sup.↓ TG                                                                       1                                                  3              A.sup.↓ AATG                                                                       1                                                  4              AA.sup.↓ AT.sup.↓ G                                                         1                                                  5              A.sup.↓ A.sup.↓ A.sup.↓ T.sup.↓                    G          1                                                  6              CGGCA       2                                                  7              CGG.sup.↓ CA                                                                       2                                                  8              C.sup.↓ GGCA                                                                       2                                                  9              CG.sup.↓ G.sup.↓ CA                                                         2                                                  10             C.sup.↓ G.sup.↓ G.sup.↓ C.sup.↓                    A          2                                                  11             UGCCA       3                                                  12             UGC.sup.↓ CA                                                                       3                                                  13             U.sup.↓ GCCA                                                                       3                                                  14             UG.sup.↓ CC.sup.↓ A                                                         3                                                  15             U.sup.↓ G.sup.↓ C.sup.↓ C.sup.↓                    A          3                                                  ______________________________________                                    

                                      TABLE 2B                                    __________________________________________________________________________                                       SEQ ID                                     OLIGOMER #                                                                            SEQUENCE                   NO:                                        __________________________________________________________________________    16      GTAAAACGACGGCCAGT          4                                          17      GTAAAACGACGGCCAG.sup.↓ T                                                                          4                                          18      GTAAAACG.sup.↓ ACGGCCAGT                                                                          4                                          19      G.sup.↓ TAAAACGACGGCCAG.sup.↓ T                                                            4                                          20      G.sup.↓ T.sup.↓ A.sup.↓ A.sup.↓                   A.sup.↓ A.sup.↓ C.sup.↓ G.sup.↓                   A.sup.↓ C.sup.↓ G.sup.↓ G.sup.↓                   C.sup.↓ C.sup.↓ A.sup.↓ G.sup.↓                                              4                                          21      GTATTCAAAGGAGTACC          5                                          22      GTATTCAAAGGAGTAC.sup.↓ C                                                                          5                                          23      GTATTCAA.sup.↓ A.sup.↓ GGAGTACC                                                            5                                          24      G.sup.↓ TATTCA.sup.↓ AAGGA.sup.↓ GTAC.sup..dw            narw. C                    5                                          25      G.sup.↓ T.sup.↓ A.sup.↓ T.sup.↓                   T.sup.↓ C.sup.↓ A.sup.↓ A.sup.↓                   A.sup.↓ G.sup.↓ G.sup.↓ A.sup.↓                   G.sup.↓ T.sup.↓ A.sup.↓ C.sup.↓                                              5                                          26      GAGCAUCACGGUGAGCG          6                                          27      GAGCAUCACGGUGAGC.sup.↓ G                                                                          6                                          28      GAGCAUCA.sup.↓ C.sup.↓ GGUGAGCG                                                            6                                          29      G.sup.↓ AGCA.sup.↓ UCACGGUG.sup.↓ AGC.sup..dw            narw. G                    6                                          30      G.sup.↓ A.sup.↓ G.sup.↓ C.sup.↓                   A.sup.↓ U.sup.↓ C.sup.↓ A.sup.↓                   C.sup.↓ G.sup.↓ G.sup.↓ U.sup.↓                   G.sup.↓ A.sup.↓ G.sup.↓ C.sup.↓                                              6                                          __________________________________________________________________________

                                      TABLE 2C                                    __________________________________________________________________________                                                   SEQ ID                         OLIGOMER #                                                                            SEQUENCE                               NO:                            __________________________________________________________________________    31      CTCTCGCACCCATCTCTCTCCTTCT              7                              32      CTCTCGCACCCATCTCTCTCCTTC.sup.↓ T                                                                              7                              33      CTCTCGCACCC.sup.↓ A.sup.↓ T.sup.↓ CTCTCTCCTTC            T                                      7                              34      CT.sup.↓ CTCGCACCCAT.sup.↓ C.sup.↓ TCTCTCCTT.            sup.↓ CT                        7                              35      C.sup.↓ T.sup.↓ C.sup.↓ T.sup.↓                   C.sup.↓ G.sup.↓ C.sup.↓ A.sup.↓                   C.sup.↓ C.sup.↓ C.sup.↓ A.sup.↓                   T.sup.↓ C.sup.↓ T.sup.↓ C.sup.↓                   T.sup.↓ C.sup.↓ T.sup.↓ C.sup.↓                   C.sup.↓ T.sup.↓ T.sup.↓ C.sup.↓                                                          7                              36      GAATGACTGATTGAGTGACTGAATG              8                              37      GAATGACTGATTGAGTGACTGAAT.sup.↓ G                                                                              8                              38      GAATGACTGA.sup.↓ T.sup.↓ TGAGTGACTGAATG                                                                8                              39      GAATGA.sup.↓ CTGA.sup.↓ T.sup.↓ TGAGTGAC.sup.            ↓ TGAAT.sup.↓ G          8                              40      G.sup.↓ A.sup.↓ A.sup.↓ T.sup.↓                   G.sup.↓ A.sup.↓ C.sup.↓ T.sup.↓                   G.sup.↓ A.sup.↓ T.sup.↓ T.sup.↓                   G.sup.↓ A.sup.↓ G.sup.↓ T.sup.↓                   G.sup.↓ A.sup.↓ C.sup.↓ T.sup.↓                   G.sup.↓ A.sup.↓ A.sup.↓ T.sup.↓                                                          8                              41      CAGUGACUGACUGAGCGACUGAACU              9                              42      CA.sup.↓ GUGACUGACUGAGCGACUGAACU                                                                              9                              43      CAGUGACUGA.sup.↓ C.sup.↓ UGAGCGACU.sup.↓                 GAACU                                  9                              44      CA.sup.↓ GUG.sup.↓ ACU.sup.↓ GACUGAGC.sup..dw            narw. G.sup.↓ ACUGAA.sup.↓ CU                                                                          9                              45      C.sup.↓ A.sup.↓ G.sup.↓ U.sup.↓                   G.sup.↓ A.sup.↓ C.sup.↓ U.sup.↓                   G.sup.↓ A.sup.↓ C.sup.↓ U.sup.↓                   G.sup.↓ A.sup.↓ G.sup.↓ C.sup.↓                   G.sup.↓ A.sup.↓ C.sup.↓ U.sup.↓                   G.sup.↓ A.sup.↓ A.sup.↓ C.sup.↓                                                          9                              __________________________________________________________________________

                                      TABLE 2D                                    __________________________________________________________________________                                                                SEQ               OLIGO-                                                      ID                MER #  SEQUENCE                                             NO:               __________________________________________________________________________    46     CGATGCGTAGGCCTAGAACTGGACTTACACTGGGATGCAAGTCAGCTGAT   10                47     CG.sup.↓ ATGCGTAGGCCTAGAACTGGACTTACACTGGGATGCAAGTCAGCTGAT                                                                   10                48     C.sup.↓ GATGCGTAG.sup.↓ GCCTAGAACTGGACT.sup.↓            TA.sup.↓ CACTGGGATGCAAGTCAGCTGAT              10                49     CG.sup.↓ ATG.sup.↓ CGTAGGCCTAGA.sup.↓                    ACT.sup.↓ G.sup.↓ GACTTACACTGGGATGCA.sup.↓               AG.sup.↓ TCAGCTGAT                            10                50     C.sup.↓ G.sup.↓ A.sup.↓ T.sup.↓                   G.sup.↓ C.sup.↓ G.sup.↓ T.sup.↓                   A.sup.↓ G.sup.↓ G.sup.↓ C.sup.↓                   C.sup.↓ T.sup.↓ A.sup.↓ G.sup.↓                   A.sup.↓ A.sup.↓ C.sup.↓ T.sup.↓                   G.sup.↓ G.sup.↓ A.sup.↓ C.sup.↓                   T.sup.↓ T.sup.↓ A.sup.↓ C.sup.↓                   A.sup.↓ C.sup.↓ T.sup.↓ G.sup.↓                   G.sup.↓ G.sup.↓                        10                       A.sup.↓ T.sup.↓ G.sup.↓ C.sup.↓                   A.sup.↓ A.sup.↓ G.sup.↓ T.sup.↓                   C.sup.↓ A.sup.↓ G.sup.↓ C.sup.↓                   T.sup.↓ G.sup.↓ A.sup.↓ T                         51     TCAGTCGAGAGCTCGAGCTCGAGCGCTAGCGATGCAGTGCGATCGATAGC   11                52     T.sup.↓ CAGTCGAGAGCTCGAGCTCGAGCGCTAGCGATGCAGTGCGATCGATAGC                                                                   11                53     TCAGTCGAGAGCTCGAGCTCGAGCGCTAGC.sup.↓ G.sup.↓                    A.sup.↓ TGC.sup.↓ AGTGCGATCGATAGC      11                54     TCAG.sup.↓ TCGAGAGC.sup.↓ TCGAGCTCGAGCGC.sup.↓            T.sup.↓ AGCGAT.sup.↓ GCAGTGCG.sup.↓ ATCGATAGC                                                                11                55     T.sup.↓ C.sup.↓ A.sup.↓ G.sup.↓                   T.sup.↓ C.sup.↓ G.sup.↓ A.sup.↓                   G.sup.↓ A.sup.↓ G.sup.↓ C.sup.↓                   T.sup.↓ C.sup.↓ G.sup.↓ A.sup.↓                   G.sup.↓ C.sup.↓ T.sup.↓ C.sup.↓                   G.sup.↓ A.sup.↓ G.sup.↓ C.sup.↓                   G.sup.↓ C.sup.↓ T.sup.↓ A.sup.↓                   G.sup.↓ C.sup.↓ G.sup.↓ A.sup.↓                   T.sup.↓ G.sup.↓                        11                       C.sup.↓ A.sup.↓ G.sup.↓ T.sup.↓                   G.sup.↓ C.sup.↓ G.sup.↓ A.sup.↓                   T.sup.↓ C.sup.↓ G.sup.↓ A.sup.↓                   T.sup.↓ A.sup.↓ G.sup.↓ C                         56     AGCGCACUAGGCAAGUCCGCGAGAUCCACGAGCCAAUACGGACUCGCAGA   12                57     AGCGCACUAGGCAAGUCCGCGAGAUCCACGAGCCAAUACGGACUCGCA.sup.↓                                                                      12                58     AGCGCACUAGGCAAGUC.sup.↓ C.sup.↓ G.sup.↓                  CGAGAUCCACGAGCCAAUACGGACUCGCAGA                      12                59     AGC.sup.↓ GCACUAGGCAA.sup.↓ GUCCGCGAGAUCCACGAGC.sup..           dwnarw. CAAUACGGA.sup.↓ C.sup.↓ UCGCAGA                                                                              12                60     A.sup.↓ G.sup.↓ C.sup.↓ G.sup.↓                   C.sup.↓ A.sup.↓ C.sup.↓ U.sup.↓                   A.sup.↓ G.sup.↓ G.sup.↓ C.sup.↓                   A.sup.↓ A.sup.↓ G.sup.↓ U.sup.↓                   C.sup.↓ C.sup.↓ G.sup.↓ C.sup.↓                   G.sup.↓ A.sup.↓ G.sup.↓ A.sup.↓                   U.sup.↓ C.sup.↓ C.sup.↓ A.sup.↓                   C.sup.↓ G.sup.↓ A.sup.↓ G.sup.↓                   C.sup.↓                                       12                       C.sup.↓ A.sup.↓ A.sup.↓ U.sup.↓                   A.sup.↓ C.sup.↓ G.sup.↓ G.sup.↓                   A.sup.↓ C.sup.↓ U.sup.↓ C.sup.↓                   G.sup.↓ C.sup.↓ A.sup.↓ G.sup.↓                   A.sup.↓                                                         __________________________________________________________________________

4. Characterization of the Functionalized Oligomers

Assessment of the oligomers is carried out by analytical ion exchangehigh pressure liquid chromatography (HPLC). For example, when oligomer17 (SEQ ID NO:4) is analyzed, a major peak is seen eluting earlier thanthat of a similar but unfunctionalized oligomer (oligomer 16, (TABLE 2)SEQ ID NO:4) with the same gradient (FIGS. 1A and 1B), confirming thatin the oligomer 17, one of the internucleoside linkages is aphosphoramidate linkage, which is non-ionic at phosphorous. Oligomers 18and 19 (both SEQ ID NO:4) which are functionalized at different sites,also show a HPLC profile similar to that of oligomer 17. Oligomer 19,which is functionalized at two sites, elutes even earlier (FIG. 1A,chromatograph (c)).

When ion exchange HPLC purified oligomer 17 is analyzed by reversedphase HPLC, it gives a doublet peak in ratio of 1:2 (FIG. 1A,chromatograph (e)) compared to oligomer 16 (FIG. 1A, chromatograph (d)).These results are due to the diastereoisomeric nature of phosphoramidateinternucleoside linkage. Similarly, oligomer 19 elutes as a broad peakbecause of two such diastereoisomeric linkages (FIG. 1F). Both oligomers17 and 19 have retention times longer than that of oligomer 16 becauseof the hydrophobic nature of the alkyl chain present in oligomers 17 and19.

Reaction of the oligomers is carried out with biotinN-hydroxysuccinimide using reported conditions (Agrawal et al. (1986)Nucl. Acid Res. 14:6227-6245). For example, the reaction mixture aftergel filtration (Sephadex G-25) of oligomer 6 shows two new peaks of thediastereomeric biotin adducts (FIG. 2A, chromatograph (b)). Similarly,reaction of oligomer (15) gives a broad peak as a doublet eluting laterthan the unreacted material (FIG. 2B, chromatographs (c) and (d).

Thus, the method described herein provides a way for functionalizing DNAand/or RNA containing oligonucleotides of any sequence andinternucleotide linkage at one or more specified sites.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AAATG5                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGGCA5                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       UGCCA5                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTAAAACGACGGCCAGT17                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTATTCAAAGGAGTACC17                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAGCAUCACGGUGAGCG17                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTCTCGCACCCATCTCTCTCCTTCT25                                                   (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GAATGACTGATTGAGTGACTGAATG25                                                   (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CAGUGACUGACUGAGCGACUGAACU25                                                   (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CGATGCGTAGGCCTAGAACTGGACTTACACTGGGATGCAAGTCAGCTGAT50                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TCAGTCGAGAGCTCGAGCTCGAGCGCTAGCGATGCAGTGCGATCGATAGC50                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AGCGCACUAGGCAAGUCCGCGAGAUCCACGAGCCAAUACGGACUCGCAGA50                          __________________________________________________________________________

What is claimed is:
 1. A compound consisting of a range of five to fiftycovalently linked nucleosides wherein at least one linkage between thenucleosides is through an aminoalkylphosphotriester residue having thestructure: ##STR7## wherein n is 2 to 6, and Nu₁ and Nu₂ are thosenucleosides adjacent said residue.
 2. The compound according to claim 1,wherein the nucleosides are selected from the group consisting ofadenosine, thymidine, cytidine, guanoside, and uridine.